The expression of polypeptides on the surface of bacteria and bacteriophage has been pursued for several years, in part because of interest in recombinant antibody production. Many other potential applications exist, including the production of genetically-engineered whole cell adsorbents, construction of "peptide libraries", cell bound enzymes, and use as live vaccines or immunogens to generate antibodies. [See, WO92/01047 and WO93/10214.]
In bacteria, one approach to obtaining surface expressed foreign proteins has been the use of native membrane proteins as a carrier for a foreign protein. In general, most attempts to develop methods of anchoring proteins on a bacterial surface have focused on fusion of the desired recombinant polypeptide to a native protein that is normally exposed on the cell's exterior with the hope that the resulting hybrid will also be localized on the surface. However, in most cases, the foreign protein interferes with localization, and thus, the fusion protein is unable to reach the cell surface. These fusions either end up at incorrect cellular locations or become anchored in the membrane with a secreted protein domain facing the periplasm [Murphy, et al., J. Bacteriol., 172:2736 (1990)].
Francisco, et al., [Proc. Natl. Acad. Sci., 89:2713 (1992)] reported constructing a surface-expression vehicle consisting of the Ipp N-terminal targeting sequence fused to a sequence derived from ompA leaving the C-terminus exposed on the external side of the outer membrane. These fusions have been reported to export a number of heterologous proteins to the E. coli surface, including .beta.-lactomase, single-chain Fv antibody and a cellulose binding protein [WO93/10214]. In addition, Fuschs, et al., [Bio/Technology, 9:1369 (1991)] reported that a fusion between the E. coli peptidoglycan-associated lipoprotein (pal) and a lysozyme-binding single-chain Fv antibody fragment could be detected on the surface of bacteria. However, in these systems, the displayed proteins were affixed to the cell surface, and thus in order to isolate purified protein, the DNA encoding the protein must be subcloned to another system.
Systems have been developed for displaying recombinant proteins, including antigens and antibodies, on the surface of filamentous bacteriophage [see, for example, WO92/01047]. In these systems, the recombinant protein is fused to the phage coat proteins expressed by either gene III (minor coat protein) or gene VIII (major coat protein). The display phage can be selectively enriched based on the binding properties of the recombinant protein. In addition, the phage carries a vector for expression of the recombinant protein-gene III fusion allowing propagation of the display phage. One of the advantages of this system is that a large library of different proteins such as Fab or single-chain Fv antibody fragments can be displayed on the phage and selected for on the basis of their binding characteristics. One disadvantage is that the number of heterologous protein molecules displayed by the phage is low, thus complicating the selection process. Another disadvantage with phage systems, as well as current bacterial systems is that the enrichment or panning process requires a significant amount of purified binding protein, e.g., antigen, and involves repeated rounds of selection and re-amplification that may result in the isolation of recombinant proteins, e.g., single-chain antibodies, with low binding affinities.
A display system combining the benefits of bacterial display and phage display has yet to be developed. Such a system would be very desirable.
It would also be desirable to have a method that can be used for cloning and protein purification with out the need for subcloning.
It would be desirable to have a display and selection method that eliminates the need for panning and purification of binding protein.